Microbial growth enhancement from a dry film additive

ABSTRACT

A hyperbranched polymer based on one or more repeating units of an AB x  type monomer, wherein A and B are functional groups and x is greater than or equal to 2, wherein A reacts with, or substantially reacts with, B, wherein B is fractionally functionlized with a plurality of functional groups comprising a first functional group comprising a C 6 -C 30  alkyl chain attached to the repeating unit through a carbonyl group (C═O) via an ester linkage, a second functional group comprising a partially fluorinated or perfluorinated C 3 -C 20  alkyl chain attached to the repeating unit through a carbonyl group (C═O) via an ester linkage, and a third functional group comprising substantially one of a stabilized radical source attached to the repeating unit via a C 0 -C 6  tether, or a 5 to 8 member chloroamide heterocycle of carbon and nitrogen that is attached to the repeating unit via a C 2 -C 6  tether.

BACKGROUND

This application is a divisional of co-pending application Ser. No.13/926,097 filed in the name of A. Williams and J. Orlicki on Jun. 25,2013. The complete disclosure of which, in its entirety, is hereinincorporated by reference.

GOVERNMENT INTEREST

Governmental Interest—The invention described herein may bemanufactured, used and licensed by or for the U.S. Government.

FIELD OF USE

Embodiments of the present invention generally relate to additives thatpromote the growth of microorganisms.

BACKGROUND

As the realm of genetically modified microorganisms increases, thepotential applications for promoting microbial growth similarlyincrease. A large array of important products may soon be producedthrough microbial means.

Therefore, the inventors have provided improved additives that promotethe growth of microorganisms.

SUMMARY

At least some embodiments of the present invention relate to ahyperbranched polymer based on one or more repeating units of an AB_(x)type monomer, wherein A and B are functional groups and x is greaterthan or equal to 2, and wherein the A functional group reacts with, orsubstantially reacts with, the B functional group, and wherein B isfractionally functionlized with a plurality of functional groups,wherein the plurality of functional groups comprise a first functionalgroup comprising a C₆-C₃₀ alkyl chain attached to the repeating unitthrough a carbonyl group (C═O) via an ester linkage, a second functionalgroup comprising a partially fluorinated or perfluorinated C₃-C₂₀ alkylchain attached to the repeating unit through a carbonyl group (C═O) viaan ester linkage, and a third functional group comprising substantiallyone of (a) a stabilized radical source attached to the repeating unitvia a C₀-C₆ tether, wherein the stabilized radical source is a 5 or 6member heterocycle of carbon and nitrogen, which may be substituted withalkyl groups α (alpha) to the stabilized radical that stericallystabilizes the oxygen free radical, or (b) a 5 to 8 member chloroamideheterocycle of carbon and nitrogen that is attached to the repeatingunit via a C₂-C₆ tether.

At least some embodiments of the present invention relate to ahyperbranched polymer that can advantageously be used to enhancemicroorganism growth atop a substrate, wherein the hyperbranched polymeris based on one or more repeating units of the following structure:

wherein one or more of R_(A) and R_(B) is one of a bond to anotherrepeating unit or, represents the fractional functionalization of thehyperbranched polymer by a plurality of functional groups, wherein theplurality of functional groups comprise a first functional groupcomprising a C₆-C₃₀ alkyl chain attached to the repeating unit through acarbonyl group (C═O) via an ester linkage, a second functional groupcomprising a partially fluorinated or perfluorinated C₃-C₂₀ alkyl chainattached to the repeating unit through a carbonyl group (C═O) via anester linkage, and a third functional group comprising substantially oneof: (a) a stabilized radical source attached to the repeating unit via aC₀-C₆ tether, wherein the stabilized radical source is a 5 or 6 memberheterocycle of carbon and nitrogen, which may be substituted with alkylgroups α (alpha) to the stabilized radical that sterically stabilizesthe oxygen free radical, or (b) a 5 to 8 member chloroamide heterocycleof carbon and nitrogen that is attached to the repeating unit via aC₂-C₆ tether.

At least some embodiments of the present invention relate to a method ofenhancing microorganism growth atop a substrate, comprising forming ahyperbranched polymer based on one or more repeating units of thefollowing formula:

wherein one of either R_(A) or R_(B) is one of a bond to anotherrepeating unit or represents the fractional functionalization of thehyperbranched polymer by a plurality of functional groups, wherein theplurality of functional groups comprises a first functional groupcomprising a C₆-C₃₀ alkyl chain attached to the repeating unit through acarbonyl group (C═O) via an ester linkage, a second functional groupcomprising a partially fluorinated or perfluorinated C₃-C₂₀ alkyl chainattached to the repeating unit through a carbonyl group (C═O) via anester linkage, and a third functional group comprising substantially oneof: (a) a stabilized radical source attached to the repeating unit via aC₀-C₆ tether, wherein the stabilized radical source is a 5 or 6 memberheterocycle of carbon and nitrogen, which may be substituted with alkylgroups α (alpha) to the stabilized radical that sterically stabilizesthe oxygen free radical, or (b) a 5 to 8 member chloroamide heterocycleof carbon and nitrogen that is attached to the repeating unit via aC₂-C₆ tether; mixing the hyperbranched polymer with a solution to form afirst mixture; curing the first mixture to form a substrate, wherein thehyperbranched polymer spontaneously segregates to an air interface ofthe substrate; and introducing one or more microorganisms atop thesurface of the substrate, wherein the hyperbranched polymer enhancesmicroorganism growth at the air interface of the substrate.

Other and further embodiments of the invention are described in moredetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the presentinvention can be understood in detail, a more particular description ofthe invention, briefly summarized above, may be had by reference toembodiments, some of which are illustrated in the appended drawings. Itis to be noted, however, that the appended drawings illustrate onlytypical embodiments of this invention and are therefore not to beconsidered limiting of its scope, for the invention may admit to otherequally effective embodiments.

FIG. 1 depicts the synthesis of an exemplary hyperbranched polymer inaccordance with some embodiments of the present invention.

FIG. 2 depicts a method of enhancing the microorganism growth atop asubstrate in accordance with some embodiments of the present invention.

To facilitate understanding, identical reference numerals have beenused, where possible, to designate identical elements that are common tothe figures. The figures are not drawn to scale and may be simplifiedfor clarity. It is contemplated that elements and features of oneembodiment may be beneficially incorporated in other embodiments withoutfurther recitation.

DETAILED DESCRIPTION

Embodiments of the present invention include additives that promote thegrowth of microorganisms. Additives in accordance with embodimentsdescribed herein advantageously promote the growth of microorganisms ona wide variety of surfaces, including surfaces that may be unsuitablefor microorganism growth without the additive. Additives in accordancewith embodiments described herein advantageously spontaneously migrate,to the upper surface (air interface) of a substrate to infuse thesubstrate with a high concentration of the additive at the airinterface. Additives in accordance with embodiments described herein mayadvantageously be used in producing a substrate, thereby providing thesubstrate with inherent microorganism growth properties. Alternatively,additives in accordance with embodiments described herein mayadvantageously be incorporated into materials suitable for coating asubstrate. Furthermore, additives in accordance with embodimentsdescribed herein advantageously provide an increase of about 10% toabout 60% in the growth of Gram positive bacteria, Gram negativebacteria, and fungi.

Embodiments of the present invention relate to a hyperbranched polymerthat can advantageously be used to modify a substrate to enhancemicroorganism growth atop the substrate. In some embodiments thehyperbranched polymer is based on one or more repeating units of anAB_(x) type monomer. In some embodiments, A and B are functional groupsand x is greater than or equal to 2. In some embodiments, the Afunctional group reacts with, or substantially reacts with, only the Bfunctional group. In some embodiments, the A functional group can be oneof, for example, an acid, an amine, a thiol, or a terminal alkyne. Insome embodiments, the B functional group can be one of, for example, analcohol, an acrylate or methacrylate (Michael addition partner), or avinyl group, or an azide. It is appreciated that the class of reactivegroups that comprise “click” chemistry partners would be especiallysuitable for this approach, whereby efficient and orthogonal (A+B)coupling pairs have been identified. Some examples were presented above(thiol-ene, alkyne-azide), but the list is illustrative only and is notexhaustive nor limiting.

In some embodiments, B is fractionally functionlized with a plurality offunctional groups described below. One exemplary structure obtained fromthe polymerization of an AB_(x) type monomer and its specificfunctionalization with a plurality of functional groups is as follows:

The structure above shows a repeat unit of the hyperbranched polymerwith two functional groups, R_(A) and R_(B). The particle bonded to therepeat unit at the left represents either the focal point of apseudo-dendron or a bond to a core molecule.

In some embodiments, one or more of R_(A) and R_(B) is one of a bond toanother repeating unit or, represents the fractional functionalizationof the hyperbranched polymer by a plurality of functional groups. Theplurality of functional groups comprises a first functional group, asecond functional group, and a third functional group described below,and modified onto the polymer chain end groups detailed herein.

In some embodiments, the first functional group is a C₆-C₃₀ alkyl chainattached to the repeating unit through a carbonyl group (C═O) via anester linkage. For example, the first functional group may be an alkylester having the formula (C═O)C_(n)H_(2n+1). In some embodiments, thefirst functional group is an aliphatic ester.

In some embodiments, the second functional group is a partiallyfluorinated or perfluorinated C₃-C₂₀ alkyl chain attached to therepeating unit through a carbonyl group (C═O). For example the secondfunctional group may be an alkyl ester having the formula(C═O)C_(n)F_(2n+1), or (C═O)CH₂C_(n)F_(2n+1). It is recognized thatother linkages may be employed to attach functional groups to thehyperbranched polymer core, dictated by the relative chemistries of boththe polymer core and the desired functional group. Linkages in additionto the shown esters include but are not limited to ethers, urethanes,amines, amides, ureas, and siloxanes.

In some embodiments, the first functional group is lauric ester havingthe formula —(C═O)(CH₂)₁₀CH₃. The lauric ester group advantageouslyincreases the solubility of the repeating unit above in most organicsolvents. In some embodiments, the second functional goup isperfluorinated octyl-ester having the formula —(C═O)(CF₂)₆CF₃. Theperfluorinated octyl-ester advantageously allows the repeating unitabove to segregate to the surface of a substrate or film. In the mostadvantageous embodiment, R_(A) is a mixture of both aliphatic ester andperfluorinated octyl-ester to maximize both solubility and propensity tosurface segregate. The incorporation of the lauric ester group and theperfluorinated octyl-ester is described in U.S. Pat. No. 7,560,520,incorporated herein by reference.

In some embodiments the third functional group is a stabilized radicalsource. In some embodiments, the stabilized radical source is, forexample (2,2,6,6-Tetramethyl-1-piperidinyloxy, free radical (TEMPO) oran N-proxyl radical. In some embodiments, the stabilized radical sourcecan be attached to the repeating unit through a C₀-C₆ tether. In someembodiments, the stabilized radical source incorporates a 5 or 6 memberheterocycle of carbon and nitrogen, which may be substituted with alkylgroups α (alpha) to the stabilized radical that sterically stabilizesthe oxygen free radical. In embodiments where the hyperbranched polymercomprises substantially the stabilized radical source, the hyperbranchedpolymer is referred to herein as a free radical enhancer.

In some embodiments, the third functional group is a3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical (referedto as 3-carboxy-proxyl free radical) having the formula C₉H₁₅NO₂. The3-carboxy-proxyl free radical advantageously promotes the growth ofbacterial microorganisms, broadly including classes of organisms such asgram-positive, gram-negative, and fungal organisms. Specific examplesinclude E. coli, S. aureus, MRSA, and C. albicans.

Alternatively, in some embodiments the third functional group is a 5 to8 member chloroamide heterocycle of carbon and nitrogen that is attachedto the repeating unit via a C₂-C₆ tether. In some embodiments, the thirdfunctional group is dichloro-s-triazinetrione (referred to asdichloroisocyanurate,) having the formula C₃Cl₂N₃O₃. It is recognizedthat this structure stabilizes oxidative chlorine species, but in thisinstance the dichloroisocyanurate advantageously leads to the growth ofbacterial microorganisms such as E. coli, or, or S. aureus, or the like.In embodiments where the third functional group is a 5 to 8 memberchloramide heterocycle attached to the repeating unit via a C₂-C₆tether, the hyperbranched polymer is referred to herein as aN-chloroamide enhancer.

In some embodiments, the hyperbranched polymer comprises about 10 mole %to about 30 mole % of the lauric ester, about 10 mole % to about 25 mole% of the perfluorinated octyl-ester group, and about 50 mole % to about75 mole % of the 3-carboxy-proxyl free radical. In some embodiments, thehyperbranched polymer comprises about 10 mole % to about 30 mole % ofthe lauric ester, about 10 mole % to about 25 mole % of theperfluorinated octyl-ester group, and about 50 mole % to about 75 mole %of the dichloroisocyanurate group.

FIG. 1 depicts an exemplary synthesis route for a hyperbranched polymerin accordance with some embodiments of the present invention. Othersynthesis routes may be available to persons of ordinary skill in theart to produce the hyperbranched polymer structures depicted in FIG. 1.As depicted in FIG. 1, a first hyperbranched polymer 100 is mixed withlauric acid 102 and perfluorinated octanoic acid 104, resulting in aintermediate hyperbranched polymer 106. As depicted in FIG. 1, the chainends, R¹, in the intermediate 106 comprise about 60 mole % hydrogen 108,about 20 mole % lauric ester 110, and about 20 mole % perfluorinatedoctyl-ester 112.

In order to synthesize the free radical enhancer, the intermediate 106is mixed with dicyclohexylcarbodiimide (DCC) and hydroxy benzotriazole(HOBt) to form the hyperbranched polymer 114 having the one or morerepeating units depicted above. As described above, the hyperbranchedpolymer 114 comprises chain ends, R², representing about 20 mole %lauric ester 110, about 20 mole % perfluorinated octyl-ester 112, andabout 60 mole % carboxy-proxyl free radical 116.

Alternatively, in order to synthesize the N-chloroamide enhancerintermediate 106, is reacted with chloroacetyl chloride, triethylamine,dichloromethane, and nitrogen (N2) to form intermediate 118. As depictedin FIG. 1, the chain ends, R³, in intermediate 118 represent about 20mole % lauric ester 110, about 20 mole % perfluorinated octyl-ester 112,and about 60 mole % chloromethylene 120. Intermediate 118 is thenreacted with sodium dichloroisocyanurate 122 to form the hyperbranchedpolymer 124 having one or more repeating units as depicted above. Asdescribed above, the hyperbranched polymer 124 comprises chain ends, R⁴,representing about 20 mole % lauric ester 110, about 20 moleperfluorinated octyl-ester 112, and about 60 mole % dichloroisocyanurate126.

FIG. 2 depicts a method of enhancing the bacterial growth atop asubstrate in accordance with some embodiments of the present invention.In some embodiments, the method 200 of enhancing bacterial growth atop asubstrate begins at 202 by forming the hyperbranched polymer, either thefree radical enhancer or the N-chloroamide enhancer, as described above.Next, at 204, the hyperbranched polymer is mixed with a polymer solutionto form a first mixture. In some embodiments, the polymer solution canbe a wide variety of polymers including but not limited to polyurethane(e.g. estane), styrinics (e.g. polystyrene), acrylics (e.g.polymethylmethacrylate, latexes), engineering plastics (e.g.polycarbonate), epoxy, reactive urethane or the like. The polymersolution is dissolved in a solvent, such as tetrahydrofuran (THF), ormethylene chloride, or ketone (e.g. acetone, methyl ethyl ketone), andincluding systems dispersed in water or fully aqueous soluble(e.g. in awater dispersible polyurethane substrate). Next, at 206, the firstmixture is cured to allow the solvent to evaporate resulting in apolymer film comprising the hyperbranched polymer. As the solventevaporates the presence of the perfluorinated octyl-ester group resultsin the segregation of the hyperbranched polymer to the surface of thepolymer film. Next, at 208, one or more microorganisms are introducedatop the surface of the polymer film, wherein the hyperbranched polymerenhances microorganism growth at the surface of the polymer film.Alternatively, in some embodiments, the hyperbranched polymer may bemixed with a soluble material such as paint, polymer, or polymerprecursors and applied to the surface of a substrate

The inventors have observed that the hyperbranched polymer unexpectedlyenhances the growth of bacterial microorganisms. While a typicaladditive with highly efficient antimicrobial function would reducemicrobial growth by about 90% to about 99% , in the current instance,the hyperbranched polymer caused the improved growth of all organismsdeposited on the substrate surface. For example, the impact on growthrates for Gram (+), Gram (−), and C. albicans organisms of about 2%additive in a thermoplastic polyurethane film is shown below in Table 1.

Growth Additive Average CFU Increase Organism Gram N- Free Organism TypeIndicator Chloramide Radical Staphylococcus aureus Bacteria + 53  60Methicillin-resistant Bacteria + 18 Not tested Staphylococcus aureusEscherichia coli Bacteria − 61 630 Candida albicans Fungi n/a 34 Nottested *as compared with inoculated controls (1 × 10⁶ Colony FormingUnits (CFU)/sample)

While the foregoing is directed to embodiments of the present invention,other and further embodiments of the invention may be devised withoutdeparting from the basic scope thereof.

1. A hyperbranched polymer based on one or more repeating units of an AB_(x) type monomer, wherein A and B are functional groups and x is greater than or equal to 2, and wherein the A functional group reacts with the B functional group, and wherein B is fractionally functionlized with a plurality of functional groups, wherein the plurality of functional groups comprise forming a hyperbranched polymer based on one or more repeating units of the following formula:

wherein one or more of R_(A) and R_(B) is one of a bond to another repeating unit or, represents the fractional functionalization of the hyperbranched polymer by a plurality of functional groups, wherein the plurality of functional groups comprise: a first functional group comprising a C₆-C₃₀ alkyl chain attached to the repeating unit through a carbonyl group (C═O) via an ester linkage, a second functional group comprising a partially fluorinated or perfluorinated C₃-C₂₀ alkyl chain attached to the repeating unit through a carbonyl group (C═O) via an ester linkage, and a third functional group comprising a stabilized radical source attached to the repeating unit via a C₀-C₆ tether, wherein the stabilized radical source is a 5 or 6 member heterocycle of carbon and nitrogen, which may be substituted with alkyl groups a to the stabilized radical that sterically stabilizes an oxygen free radical, or a 5 to 8 member chloroamide heterocycle of carbon and nitrogen that is attached to the repeating unit via a C₂-C₆ tether.
 2. A method of enhancing microorganism growth atop a substrate using the hyperbranched polymer of claim 1, further comprising mixing the hyperbranched polymer with a solution to form a first mixture; curing the first mixture to form a substrate, wherein the hyperbranched polymer spontaneously segregates to an air interface of the substrate; and introducing one or more microorganisms atop the surface of the substrate, wherein the hyperbranched polymer enhances microorganism growth at the air interface of the substrate.
 3. The method of claim 2, wherein the first functional group is a lauric ester, the second functional group is a perfluorinated octyl-ester, and the third functional group is substantially one of a 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical or a dichloro-s-triazinetrione.
 4. The method of claim 3, wherein the hyperbranched polymer comprises about 10 mole % to about 30 mole % of the lauric ester, about 10 mole % to about 25 mole % of the perfluorinated octyl-ester, and about 50 mole % to about 75 mole % of the 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical.
 5. The method of claim 3, wherein the hyperbranched polymer comprises about 10 mole % to about 30 mole % of the lauric ester, about 10 mole % to about 25 mole % of the perfluorinated octyl-ester, and about 50 mole % to about 75 mole % of the dichloro-s-triazinetrione.
 6. The method of claim 2, wherein the substrate is a thermoplastic polyurethane film.
 7. The method of claim 2, wherein the hyperbranched polymer increases the growth of gram-positive and gram-negative bacteria about 10 percent to about 60 percent at the air interface of the substrate. 